Achieving a functional cure for HIV that allows cessation of anti-retroviral therapy (ART) will require development of specific strategies to recognize and deplete infected cell reservoirs. HIV-1 establishes a pool of latently infected, resting memory CD4+ T cells during primary infection that persist during ART treatment with and can resume active viral replication within weeks following treatment interruption. The latent reservoir is very stable with a half-life of 44 months in the presence of ART, may be replenished during intermittent viremia or by homeostatic proliferation of latently infected cells, and is not significantly diminished by ART intensification. Importantly, establishment of active and latent HIV reservoirs occurs primarily in the secondary lymphoid tissues of the body, and therefore these tissues are the central battleground for HIV immunotherapy. The mucosal immune system in the oral cavity is an extensive and compartmentalized mucosaassociated lymphoid tissue (MALT) with a specialized microanatomy and composition adapted for defense against oral pathogens. While HIV is uncommonly transmitted via the oral route, the rich lymphoid environment of the oral MALT, particularly the tonsils, makes these tissues highly susceptible to infection and establishment of HIV reservoirs. Efforts to eradicate HIV infection in patients will undoubtedly need to identify the specific populations of cells harboring latent virus in the oral cavity and devise strategies to effectively destroy these cells. To date, the majority of research into HIV latency in vivo has been derived from analysis of circulating quiescent T cells, which differ significantly from lymphoid tissues that support the majority of viral replication in infectd individuals and serve as the initial seeding ground for latent founder virus populations. The overarching goal of this application is to define cells in the oral MALT that are susceptible to HI infection-and therefore potential reservoirs of latent infection-and to explore a novel natural killer (NK) cell approach to eradicating latently infected cells. To achieve this goal, we have established a highly collaborative team of experts in HIV virology and latency (Drs. Karn, McDonald, and Tilton) and oral mucosal immunology (Dr. Pandayan) and leveraged novel and powerful flow cytometry and imaging technologies coupled to a robust tissue explant model using ex vivo cultured tonsillar tissues. We have established innovative protocols to model latent infection in tonsillar tissues as well as methods to expand and engineer autologous NK cell populations for destruction of infected cells in the tonsils. In this application, Dr. Tilton's grop will adapt novel flow cytometry assays developed by his group to identify targets of infection and latency in the oral cavity (Aim 1). Dr. McDonald will utilize his expertise in live cell fluorescene microscopy to directly visualize those infectious events within the living lymphoid tissue (Aim 2). Anchoring the team is Dr. Karn, who has played a central role in identifying the signaling machinery responsible for entry into and reactivation from HIV latency using chromatin immunoprecipitation (ChIP) and deep sequencing technologies. The Karn group will adapt the protocols they have developed to expand and genetically modify activated NK cells that are capable of specifically killing reactivated HIV infected cells (Aims 3 & 4). The successful completion of this work will for the first time identify cellular populations in the oral cavity susceptible to productive and latent HIV-1 infection, establish biologically relevant models of latent infection in these tissues, and explore a promising and novel strategy to eradicate infected cells using activated and expanded autologous or engineered NK cells. We believe our systematic approach of characterizing the HIV infected cells, monitoring NK cell killing ex vivo and engineering NK cells with specific chimeric antigen receptors will provide unprecedented insight into oral mucosal MALT cell populations susceptible to latent infection and permit development of new and effective technologies to clear latent HIV reservoirs in the oral cavity.